Protein phosphorylation is a common regulatory mechanism used by cells to selectively modify proteins carrying regulatory signals from outside the cell to the nucleus. The proteins that execute these biochemical modifications are a group of enzymes known as protein kinases. Kinases represent a large family of enzymes with an estimated 1000 different forms present in the human genome. They play an important role in regulation of signal transduction by adding phosphate groups to various proteins in cellular signaling pathways. Depending on the phosphate accepting protein, the addition of a phosphate group may up-regulate or down regulate the signal. The protein kinases come in two general classes that are defined by the particular amino acid that is phosphorylated. These are tyrosine kinases and serine/threonine kinases.
The complementary activity of dephosphorylation is a function of another group of enzymes known as protein phosphatases. Protein phosphatases can be broadly classified into two families, protein serine/threonine (ser/thr) phosphatases (PP) and protein tyrosine (tyr) phosphatases (PTPase). Tyrosine phosphatases generally catalyze the dephosphorylation of tyrosine residues, though some have dual specificity for both phosphoser/thr and phosphotyrosine. Ser/thr phosphatases catalyze the dephosphorylation of phosphoserine and phosphothreonine residues in proteins and peptides and have been further divided into three types, PP1, PP2A and PP2B. PTP/PPases provide reversible protein phosphorylation through the dephosphorylation of phosphoamino acids and function in the control of cellular proliferation, differentiation and other cellular processes. These phosphatases can be classified into soluble or transmembrane proteins.
Kinases and phosphatases are often validated drug targets since human diseases are frequently linked to dysregulation of cellular signaling pathways. The profound cellular effects mediated by tyrosine kinases and serine kinases, for example, their putative role in angiogenesis [Giroux, S. et al. Curr. Biol. 9: 369 (1999)], lymphoid development [Nosaka, T., et al. Science 270: 800, (1995)] and insulin resistance [Yuan, M. et al., Science 293: 1673, (2001)] coupled with the implication that mutant or defective tyrosine kinase variants may be involved in tumorigenesis [Jeffers, M., et al. PNAS 94: 11445, (1997)], have made them attractive targets for the development of new therapeutic molecules.
Methods for measuring the phosphorylation state of cellular proteins have traditionally relied on radioactive means, for example, 32P or 33P-gammaphosphate incorporation or assays employing labeled antibodies. Phosphorylated tyrosine residues, for example, can be detected either by immunoprecipitation or blotting using a radiolabeled antiphosphotyrosine antibody or using filter binding to trap radiolabeled products. The fact that the techniques for detecting radioactive isotope (i.e. blotting, immunoprecipitation, gel electrophoresis) are very time consuming, however, minimizes the appeal of these methodologies for high throughput screening.
More recent methods utilize a standard enzyme-linked immunosorbent assay (ELISA) format for measuring kinase or phosphatase activity. These methods utilize purified heterologous substrate protein or synthetic substrate peptides anchored to a microtiter plate. After exposure of the substrate molecule to a sample containing the appropriate kinase or phosphatase, the level of phosphorylation is evaluated with antiphosphotyrosine antibodies to quantitate the amount of phosphorylated protein bound to the plate. In this method, kinase or phosphatase activity is determined after any unbound antibody is removed from the plate.
Hirth et al., U.S. Pat. No. 5,763,198, for example, describes an ELISA-type assay in which a substrate-specific antibody is used as an anchoring molecule to isolate a protein substrate from a cell lysate preparation and immobilize it on a solid phase support. Hirth's method then determines the level of kinase activity or phosphatase activity by evaluating the tyrosine phosphorylation state of the protein substrate bound to the solid phase. Hirth's method uses an anti-phosphotyrosine antibody as the detecting molecule and the unbound portion of the anti-phosphotyrosine is removed by a plate washing procedure.
A homogenous assay has been described for tyrosine kinases involving the use of a fluorescently labeled peptide and an antibody that is specific for the phosphorylated product. This method employs fluorescence polarization for detection where the antibody/labeled peptide complex possesses a greater polarization signal than the free labeled peptide. Other methods for measuring tyrosine kinase activity, particularly tyrosine kinase receptor activity, are described in WO95/04136, EP 0 730 740 B1, and U.S. Pat. No. 5,599,681.
The availability of an efficient, high throughput assay of kinase activity or phosphatase activity, particularly, one that does not rely on either radioactive labels or antibodies and does not require any separation steps, is highly desirable because it provides the means, not only to determine the level of kinase or phosphatase activity in a sample, but to do so without the limitations associated with the use of radioisotopes and antibodies.